4th Shifting Micro Lab Reviewer
4th Shifting Micro Lab Reviewer
4th Shifting Micro Lab Reviewer
Specimen Collection
A properly collected specimen is the single most
important step in the diagnosis of an infection.
1. Sterile collection method and devices
2. Quantity of material must be adequate, with
accurate and complete label.
3. Sample should be representative of the infectious
process.
4. Prompt transport to lab to avoid overgrowth of
fungal or bacterial contaminants.
5. Physician has suspected diagnosis for special
procedures or specimen treatment.
Specimen Collection
6. Collect the right specimen:
Mycoses Specimen
Recovery of bacteria and fungi is most significant if the agent is isolated from a
normally sterile site.
•Significant if (+): blood, CSF, joint fluid, pleural cavity
•Consider in the context of normal flora: respiratory tract, GIT, GUT, wounds
or skin
Laboratory Precautions
• Conidia/ spores remain dormant even when
aerosolized.
• Laboratory procedures must be done in an
biosafety cabinet (BSL 3)
• Remember: Inhalation is a common route of
infection.
Microscopic Identification
Procedure Remarks
Calcofluor white •Binds to cellulose and chitin in the cell walls of fungi
stain •Fluoresces under long-wavelength UV light
•May demonstrate morphology that is diagnostic of the species
•Ex. spherules with endospores in Coccidioides immitis infection
Periodic Acid Schiff •Stain tissue sections when fungal infection is suspected
(PAS) •Oxidizes hydroxyl in the cell wall carbohydrate
Gomori •Fungi appear black against a pale background
Methenamine •For screening of clinical speciments
Silver Stain (GMS) •Identify Pneumocystis jiroveci cysts
Giemsa, Wright’s •Detect intracellular Histoplasma capsulatum in blood smears, lymph nodes, lung, liver,
stain bone marrow
•Cell light to dark blue in color
Lactophenol cotton •Stain used to distinguish fungal growth and identify organisms by morphology
blue (LPCB) •Used in tease preparation
India ink, Nigrosin •Used to identify Cryptococcus neoformans capsule
Microscopic Identification
Coccidioidomycosis
Histoplasmosis
Blastomycosis
Paracoccidiomycosis
Systemic mycoses
• Caused by dimorphic fungi
Pulmonary
Inhalation DIssemination
infection
Etiologic Agent:
Coccidioides immitis
Colonial morphology
– Hyphae,barrel-shaped, arthroconidia
Clinical findings
Primary Infection:
- Asymptomatic in most
- Nodular lesions in lungs
Secondary (disseminated)
infection (1%):
- Chronic/fulminant
- Infection of lungs,meninges,
bones and skin
Diagnosis
• Etiologic agent:
– Histoplasma capsulatum
- 70% of patients
- Haematogenous spread
- Painless
- Verrucous lesions with
irregular borders , or as
ulcers
- Most Frequent Sites: Face,
upper limbs, neck and scalp
Paracoccidiomycosis
• Etiologic agent:
– Paracoccidioides brasiliensis
• Ulcerated lesion
in pharyngeal
mucosa
• Extensive
destruction of
facial features
Penicilliosis marneffei
Pityriasis versicolor
Keratomycosis
Tinea nigra
Black piedra
White piedra
Superficial Mycoses
• Limited to the outmost layer of skin and hair
Pityriasis (Tinea) versicolor
Etiologic Agent:
Malassezia furfur complex
M. globosa
M. restricta
Pityriasis (Tinea) versicolor
Characteristic Lesion:
Discrete, serpentine, hyper or hypopigmented macules occurring on
the skin of the chest, upper back, arms and abdomen
Wood’s lamp observation of lesions
(+) = Yellow Fluorescence
(positive yang pictures ah haha)
M. furfur Morphology
“Spaghetti and Meatballs Appearance”
M. furfur Morphology
Broadly budding yeast cells
Bottle shaped with a collarette-like
thickening seen at the junction of the mother and
daughter cells
Short hyphae, yeast cells
Etiologic Agent:
Saprophytic fungi
Aspergillus
Fusarium
Alternaria
Candida
Histoplasma capsulatum
(+)corneal ulcers
Tinea Nigra
Etiologic Agent:
Exophiala werneckii (Hortaea
werneckii)
Tinea Nigra
Lesions appear as a
dark (brown to black)
discoloration, often
on the palm (d/t
melanin produced by
the fungi)
Brownish maculae
on palms, fingers,
face, soles of feet
E. werneckii Morphology
Branched, septate hyphae and budding yeast
cells with melaninized cell walls (brown in
color)
E. werneckii Morphology
Branched, septate hyphae and budding yeast
cells with melaninized cell walls (brown in
color)
E. werneckii Morphology
Branched, septate hyphae and budding yeast
cells with melaninized cell walls (brown in
color)
E. werneckii Morphology
Branched, septate hyphae and budding yeast
cells with melaninized cell walls (brown in
color)
E. werneckii Culture
Black colonies
on culture
medium
Black piedra
Etiologic Agent:
Piedraia hortae
Black piedra
Etiologic Agent:
Trichosporon beigelii
White piedra
Soft, white to yellowish nodules loosely
attached to the hair
Occur as a sleeve or collarette around the
hair shaft
T. beigelii Morphology
Intertwined septate hyphae
Blastoconidia and arthroconidia
T. beigelii Morphology
Intertwined septate hyphae
Blastoconidia and arthroconidia
T. beigelii Culture
Dermatophytoses
Trichophyton spp.
Microsporum spp.
Epidermophyton spp.
Dermatophytes
In dealing with dermatophyte identification, we
need to consider two things: microconidia and
macroconidia. If you know the feature
associated with a specific dermatophyte
genus, G ka na bes! Since species within a
genus have almost the same features
Trichophyton spp.
Thin-walled
macroconidia
Teardrop
microconidia
Macroconidium: Fusiform
Present multinucleated with thick rough walls
MACROCONIDIA
HYPHAE
MICROCONIDIA
Microsporum spp.
Fusiform
macroconidia
In M. canis,
macroconidia may be
barrel-shaped
Thick-walled
macroconidia
Few oval
microconidia
MACROCONIDIA
HYPHAE
MICROCONIDIA
Microsporum spp.Culture
Macroconidium: Clavate/Clubshaped
Present with 3-5 cells and thin smooth walls
Cluster in groups of 3 or 4
Chlamydoconidia typically present in older cultures
Colonies are usually flat and velvety with a tan to olive-green
tinge
Microconidium: ABSENT
Epidermophyton floccosum
Epidermophyton floccosum
MACROCONIDIA
HYPHAE
Epidermophyton floccosum
Thin smooth walled
macroconidia
NO MACROCONIDIA
MICROCONIDIA
HYPHAE
E. Flocossum Culture
• Tinea barbae
• Tinea corporis
• Tinea capitis
• Tinea cruris
• Tinea pedis
• Tinea manuum
• Tinea unguium
Tinea barbae (beard)
Gray-patchringworm
M. audouini/canis
Black-dotringworm Fungatingexophytic masses(kerions)
T. tonsurans T. tonsurans,T. schoenleini, T.violaceum
Tinea cruris (groin)
E. floccossum
Tinea pedis (foot)
Mocassin
Vesicular
T. mentagrophytes
T. rubrum
Interdigital
E. flocossum
Tinea manuum (Hand)
Tinea unguium (Nails)
T. mentagrophytes
T. rubrum
E. flocossum
• Hyphae: Narrow
hyaline septate
• Conidia: Abundant,
oval borne on delicate
• Or in rosette or daisy petal
conidophores
Sporothrix schenckii (Yeast)
• Spherica, oval or;
• Elongated (“Cigar-shaped”)
• With single or (rarely) multiple
buds
Sporothrix schenckii
• Asteroid body
– Often seen in tissue
– In H&E, consists of a
central basophilic yeast
cell
– Surrounded by
radiating extension of
eosinophilic material, w/c
are
depositions of Ag-Ab
complexes
and complement
Sporothrix schenckii
Asteroid body
• The spheric yeastlike cells
are surrounded by
Splendore-Hoeepli material
Chromoblastomycosis
• Caused by dematiaceous fungi
(fungi that have brown to black
melanin pigments in their cell
wall
• Wart-like
• Cauliflower-like
• With abscesses covering the
area
• With covering the warty
surface
“black dots”
Chromoblastomycosis
• Molds are morphologically diverse, and most
are capable of producing several different
forms when grown in culture.
1. Phialophora verrucosa
2. Cladosporium carrionii
3. Rhinocladiella aquaspersa
4. Fonsecaea pedrosoi
5. Fonsecaea compacta
Phialophora verrucosa
• Conidia produced
from
flask-shaped
phialides
with cup-shaped
collarettes
• Conidia (mature):
spherical to oval,
extruded from the
phialide and
accumulate
around it
Cladosporium carrionii
• Conidia: branching chains
by distal (acropetalous)
budding
• Elongated conidiophores
with long,branching chains
of oval conidia
• Species identified based on
differences in the length of
the chains and shape and
size of the conidia
Fonsecaea pedrosoi
• Polymorphic phialides
with
chains of blastoconidia
similar to Cladosporium
sp
• Sympodial,rhinocladiella
type of condition
• Most strains form short
branching chains of
blastoconidia as well as
sympodial conidia
Fonsecaea compacta
• Blastoconidia: almost
spherical, with broad
base connecting the
condiia
• Structures are smaller
and more compact than
those of F.pedrosoi
Rhinocladiella aquaspersa
• Conidia: lateral or
terminal, from a
lengthening
condigenous
cell (Sympodial
process)
– Elliptical to
clavate
shaped
Chromoblastomycosis
• In tissue, the fungi that cause
chromoblastomycosis ALL characteristically
form murifom cells (sclerotic bodies, Medlar
bodies) that are chestnut brown because of
the melanin in their walls
Medlar body
• Brown-pigmented murifom
cell of chromoblastomycosis
Mycetoma
• Atinomycetoma- caused by actinomycete
– More invasive
• Eumycetoma- caused by a fungus
– Pseudoallescheria boydii
– Madurella mycetomatis
Exophiala jeanselmei
• Dimorphic fungi with
brown or black color or
dry aspect
• Dark yeast cells, septate,
and branched hyphae
• Unicellular anneloconidias
accumulates themselves in
the apex of elongated annelids
and with terminal or intercalate
formation
Curvalaria geniculata
• Mycetoma granule
Hard,contain intertwined,
septate hyphae that are
typically distorted and
enlarged
at the periphery of the
granule
Curvularia geniculata
• Myetoma granule
hyphage may be
embedded in an
amorphous cement like
substance Splendore-
Hoeppli
materials which
interdigitates
at the periphery of the
granules
Phaeohypomycosis
• Term used to describe a
heterogeneous array of
fungal infections caused
by pigmented, or
dematiaceous, fungi
which are present in
tissue as irregular hyphae
rather than the sclerotic
muriform cells seen in
chromoblastomycosis
Phaeohypomycosis
Opportunistic Mycoses
• Includes:
o Endogenous opportunists – Candida (Candidiasis)
o Exogenous opportunists
Cryptococcosis
Aspergillosis
Mucormycosis
Pneumocystis pneumonia
Penicilliosis
Candidiasis
• members of the normal flora of the skin,
mucous membranes, and gastrointestinal
tract.
• most common systemic mycosis
• In tissue, (37 C)
o germ tubes
• In the environment (25 C)
o Blastoconidia, pseudohyphae, and
chlamydoconidia (chlamydospores)
Candidiasis
• Culture
From Murray:
CHROMAGAR
• Differentiation of Candida species
• The green colonies are C. albicans, the blue-gray
colonies are C. tropicalis, and the large, rough,
pale pink colony is C. krusei.
• The smooth, pink or mauve colonies are another
yeast species (only C. albicans, C. tropicalis, and
C. krusei can be reliably recognized on this
media; other species have colonies ranging from
white, to pink, to mauve
Candidiasis
• In culture,
Cryptococcus species
produce whitish
mucoid colonies
within 2–3 days.
Cryptococcosis
• Microscopically, in
culture or clinical
material, the spherical
budding yeast cells (5–10
μm in diameter) are
surrounded by a thick
nonstaining capsule
Cryptococcosis
Yeast cell
with thick
capsule
unstained by
India ink
• In tissue and upon staining
with India ink, the cells are
variable in size, spheric,
oval, or elliptic, and are From CDC: C. neoformans stained with India Ink
surrounded by optically
clear, smoothly contoured,
spheric zones or “halos”
that represent the
extracellular polysaccharide
capsule
• Ubiquitous in nature
• A. fumigatus is most common human
pathogen
• Produces abundant small conidia that are
easily aerosolized
• In immunocompromised patients, the conidia
may germinate to produce hyphae that invade
the lungs and other tissues
• In the environment (25 C), long conidiophores
with terminal vesicles on which phialides
produce basipetal chains of conidia
From CDC
• Grow in culture as hyaline molds.
• On a gross level, the colonies of
Aspergillus may be black, brown, green,
yellow, white, or other colors, depending
upon the species and the growth
conditions (Murray)
• On direct examination of
sputum with KOH or
calcofluor white or in
histologic sections, the
hyphae of Aspergillus
species are hyaline,
septate, and uniform in
width (about 4 μm) and
branch dichotomously
(Jawetz)
Mucormycosis
• Mucormycosis (zygomycosis) is
an opportunistic mycosis
caused by a number of molds
classified in the order
Mucorales of the Phylum
Glomerulomycota and
Subphylum Mucoromycotina.
• These fungi grow rapidly on
laboratory media, producing
abundant cottony colonies.
Mucormycosis
Cunninghamela sp.
Mucormycosis
On Sabouraud's dextrose
agar colonies are colonies are
very fast growing, cottony to
fluffy, white to yellow,
becoming dark-grey, with the
development of sporangia.
Mucormycosis (Rhinocerebral infection)
• Direct examination or culture of
nasal discharge, tissue, or sputum
will reveal broad hyphae (10–15
μm) with uneven thickness,
irregular branching, and sparse
septations
Pneumocystis jirovicii
• Causes pneumonia in immunocompromised patients
• P. jirovicii is the human species
• P. carinii is found only in rats
CDC: Cysts in the smear of bronchoalveolar CDC: Histopathology showing Pneumocystis cysts
lavage material in the lung of a patient with AIDS
Pneumocystis jirovicii
• Thin-walled trophozoites and cysts,
which are thick-walled, spherical to
elliptical (4–6 μm), and contain four to
eight nuclei
• Cysts can be stained with silver stain,
toluidine blue, and calcofluor white
• Trophozoites and cysts are present in a
tight mass that probably reflects their
mode of growth in the host
• Contains a surface glycoprotein that can
be detected in sera of acutely ill or
normal individuals